Inbred maize line PH6WR

ABSTRACT

An inbred maize line, designated PH6WR, the plants and seeds of inbred maize line PH6WR, methods for producing a maize plant, either inbred or hybrid, produced by crossing the inbred maize line PH6WR with itself or with another maize plant, and hybrid maize seeds and plants produced by crossing the inbred line PH6WR with another maize line or plant and to methods for producing a maize plant containing in its genetic material one or more transgenes and to the transgenic maize plants produced by that method. This invention also relates to inbred maize lines derived from inbred maize line PH6WR, to methods for producing other inbred maize lines derived from inbred maize line PH6WR and to the inbred maize lines derived by the use of those methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/758,858 filed Jan. 11, 2001, now U.S. Pat. No. 6,717,038, thecontents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention is in the field of maize breeding, specifically relatingto an inbred maize line designated PH6WR.

BACKGROUND OF THE INVENTION

The goal of plant breeding is to combine in a single variety or hybridvarious desirable traits. For field crops, these traits may includeresistance to diseases and insects, tolerance to heat and drought,reducing the time to crop maturity, greater yield, and better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination and stand establishment, growthrate, maturity, and plant and ear height, is important.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Maize (zea mays L.), often referred to as corn in the United States, canbe bred by both self-pollination and cross-pollination techniques. Maizehas separate male and female flowers on the same plant, located on thetassel and the ear, respectively. Natural pollination occurs in maizewhen wind blows pollen from the tassels to the silks that protrude fromthe tops of the ears.

A reliable method of controlling male fertility in plants offers theopportunity for improved plant breeding. This is especially true fordevelopment of maize hybrids, which relies upon some sort of malesterility system. There are several options for controlling malefertility available to breeders, such as: manual or mechanicalemasculation (or detasseling), cytoplasmic male sterility, genetic malesterility, gametocides and the like.

Hybrid maize seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent inmaize plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile maize and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., of Pioneer Hi-Bred, U.S. Pat. No.5,432,068, have developed a system of nuclear male sterility whichincludes: identifying a gene which is critical to male fertility;silencing this native gene which is critical to male fertility; removingthe native promoter from the essential male fertility gene and replacingit with an inducible promoter; inserting this genetically engineeredgene back into the plant; and thus creating a plant that is male sterilebecause the inducible promoter is not “on” resulting in the malefertility gene not being transcribed. Fertility is restored by inducing,or turning “on”, the promoter, which in turn allows the gene thatconfers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran antisense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see:Fabinjanski, et al. EPO 89/3010153.8 Publication No. 329,308 and PCTApplication PCT/CA90/00037 published as WO 90/08828).

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, Glenn R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach.

Development of Maize Inbred Lines

The use of male sterile inbreds is but one factor in the production ofmaize hybrids. Plant breeding techniques known in the art and used in amaize plant breeding program include, but are not limited to, recurrentselection, backcrossing, pedigree breeding, restriction lengthpolymorphism enhanced selection, genetic marker enhanced selection andtransformation. The development of maize hybrids in a maize plantbreeding program requires, in general, the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Pedigree breeding and recurrent selection breeding methods areused to develop inbred lines from breeding populations. Maize plantbreeding programs combine the genetic backgrounds from two or moreinbred lines or various other germplasm sources into breeding pools fromwhich new inbred lines are developed by selfing and selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which of thosehave commercial potential. Plant breeding and hybrid development, aspracticed in a maize plant breeding program, are expensive and timeconsuming processes.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding five or more generations ofselfing and selection is practiced: F₁→F₂; F₂→F₃; F₃→F₄; F₄→F₅, etc.

Recurrent selection breeding, backcrossing for example, can be used toimprove an inbred line and a hybrid which is made using those inbreds.Backcrossing can be used to transfer a specific desirable trait from oneinbred or source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred(recurrent parent) to a donor inbred (non-recurrent parent), thatcarries the appropriate gene(s) for the trait in question. The progenyof this cross is then mated back to the superior recurrent parentfollowed by selection in the resultant progeny for the desired trait tobe transferred from the non-recurrent parent. After five or morebackcross generations with selection for the desired trait, the progenywill be homozygous for loci controlling the characteristic beingtransferred, but will be like the superior parent for essentially allother genes. The last backcross generation is then selfed to give purebreeding progeny for the gene(s) being transferred. A hybrid developedfrom inbreds containing the transferred gene(s) is essentially the sameas a hybrid developed from the same inbreds without the transferredgene(s).

Elite inbred lines, that is, pure breeding, homozygous inbred lines, canalso be used as starting materials for breeding or source populationsfrom which to develop other inbred lines. These inbred lines derivedfrom elite inbred lines can be developed using the pedigree breeding andrecurrent selection breeding methods described earlier. As an example,when backcross breeding is used to create these derived lines in a maizeplant breeding program, elite inbreds can be used as a parental line orstarting material or source population and can serve as either the donoror recurrent parent.

Development of Maize Hybrids

A single cross maize hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F₁. In thedevelopment of commercial hybrids in a maize plant breeding program,only the F₁ hybrid plants are sought. Preferred F₁ hybrids are morevigorous than their inbred parents. This hybrid vigor, or heterosis, canbe manifested in many polygenic traits, including increased vegetativegrowth and increased yield.

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, although different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrid progeny (F₁). Duringthe inbreeding process in maize, the vigor of the lines decreases. Vigoris restored when two different inbred lines are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred lines is that the hybrid between a definedpair of inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

A single cross hybrid is produced when two inbred lines are crossed toproduce the F₁ progeny. A double cross hybrid is produced from fourinbred lines (or synthetics) crossed in pairs (A×B and C×D) and then thetwo F₁ hybrids are crossed again (A×B)×(C×D). A three-way cross hybridis produced from three inbred lines (or synthetics) where two of theinbred lines (or synthetics) are crossed (A×B) and then the resulting F₁hybrid is crossed with the third inbred (or synthetics) (A×B)×C. Much ofthe hybrid vigor exhibited by F₁ hybrids is lost in the next generation(F₂). Consequently, seed from hybrids is not used for planting stock.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female parent. Incomplete removal or inactivation of thepollen provides the potential for self pollination. This inadvertentlyself pollinated seed may be unintentionally harvested and packaged withhybrid seed.

Once the seed is planted, it is possible to identify and select theseself pollinated plants. These self pollinated plants will be geneticallyequivalent to the female inbred line used to produce the hybrid.

Typically these self pollinated plants can be identified and selecteddue to their decreased vigor. Female selfs are identified by their lessvigorous appearance for vegetative and/or reproductive characteristics,including shorter plant height, small ear size, ear and kernel shape,cob color, or other characteristics.

Identification of these self-pollinated lines can also be accomplishedthrough molecular marker analyses. See, “The Identification of FemaleSelfs in Hybrid Maize: A Comparison Using Electrophoresis andMorphology”, Smith, J. S. C. and Wych, R. D., Seed Science andTechnology 14, pp. 1–8 (1995), the disclosure of which is expresslyincorporated herein by reference. Through these technologies, thehomozygosity of the self pollinated line can be verified by analyzingallelic composition at various loci along the genome. Those methodsallow for rapid identification of the invention disclosed herein. Seealso, “Identification of Atypical Plants in Hybrid Maize Seed byPostcontrol and Electrophoresis” Sarca, V. et al., Probleme de GeneticaTeoritica si Aplicata Vol. 20 (1) p. 29–42.

As is readily apparent to one skilled in the art, the foregoing are onlysome of the various ways by which the inbred can be obtained by thoselooking to use the germplasm. Other means are available, and the aboveexamples are illustrative only.

Maize is an important and valuable field crop. Thus, a continuing goalof plant breeders is to develop high-yielding maize hybrids that areagronomically sound based on stable inbred lines. The reasons for thisgoal are obvious: to maximize the amount of grain produced with theinputs used and minimize susceptibility of the crop to pests andenvironmental stresses. To accomplish this goal, the maize breeder mustselect and develop superior inbred parental lines for producing hybrids.This requires identification and selection of genetically uniqueindividuals that occur in a segregating population. The segregatingpopulation is the result of a combination of crossover events plus theindependent assortment of specific combinations of alleles at many geneloci that results in specific genotypes. The probability of selectingany one individual with a specific genotype from a breeding cross isinfinitesimal due to the large number of segregating genes and theunlimited recombinations of these genes, some of which may be closelylinked. However, the genetic variation among individual progeny of abreeding cross allows for the identification of rare and valuable newgenotypes. These new genotypes are neither predictable nor incrementalin value, but rather the result of manifested genetic variation combinedwith selection methods, environments and the actions of the breeder.

Thus, even if the entire genotypes of the parents of the breeding crosswere characterized and a desired genotype known, only a few if anyindividuals having the desired genotype may be found in a largesegregating F₂ population. Typically, however, neither the genotypes ofthe breeding cross parents nor the desired genotype to be selected isknown in any detail. In addition to the preceding problem, it is notknown how the genotype would react with the environment. This genotypeby environment interaction is an important, yet unpredictable, factor inplant breeding. A breeder of ordinary skill in the art cannot predictthe genotype, how that genotype will interact with various climaticconditions or the resulting phenotypes of the developing lines, exceptperhaps in a very broad and general fashion. A breeder of ordinary skillin the art would also be unable to recreate the same line twice from thevery same original parents as the breeder is unable to direct how thegenomes combine or how they will interact with the environmentalconditions. This unpredictability results in the expenditure of largeamounts of research resources in the development of a superior new maizeinbred line.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred maize line,designated PH6WR. This invention thus relates to the seeds of inbredmaize line PH6WR, to the plants of inbred maize line PH6WR, to methodsfor producing a maize plant produced by crossing the inbred maize linePH6WR with itself or another maize line, and to methods for producing amaize plant containing in its genetic material one or more transgenesand to the transgenic maize plants produced by that method. Thisinvention also relates to inbred maize lines derived from inbred maizeline PH6WR, to methods for producing other inbred maize lines derivedfrom inbred maize line PH6WR and to the inbred maize lines derived bythe use of those methods. This invention further relates to hybrid maizeseeds and plants produced by crossing the inbred line PH6WR with anothermaize line.

DEFINITIONS

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. NOTE: ABS is in absolute termsand % MN is percent of the mean for the experiments in which the inbredor hybrid was grown. These designators will follow the descriptors todenote how the values are to be interpreted. Below are the descriptorsused in the data tables included herein.

ABTSTK=ARTIFICIAL BRITTLE STALK. A count of the number of “snapped”plants per plot following machine snapping. A snapped plant has itsstalk completely snapped at a node between the base of the plant and thenode above the ear. Expressed as percent of plants that did not snap.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola). A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher score indicates a higher resistance.

BAR PLT=BARREN PLANTS. The percent of plants per plot that were notbarren (lack ears).

BRT STK=BRITTLE STALKS. This is a measure of the stalk breakage near thetime of pollination, and is an indication of whether a hybrid or inbredwould snap or break near the time of flowering under severe winds. Dataare presented as percentage of plants that did not snap.

BU ACR=YIELD (BUSHELS/ACRE). Yield of the grain at harvest in bushelsper acre adjusted to 15.5% moisture.

CLD TST=COLD TEST. The percent of plants that germinate under cold testconditions.

CLN=CORN LETHAL NECROSIS. Synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance.

COM RST=COMMON RUST (Puccinia sorghi). A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance.

D/D=DRYDOWN. This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1–9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

DIP ERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora). A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance.

DIPROT=DIPLODIA STALK ROT SCORE. Score of stalk rot severity due toDiplodia (Diplodia maydis). Expressed as a 1 to 9 score with 9 beinghighly resistant.

DRP EAR=DROPPED EARS. A measure of the number of dropped ears per plotand represents the percentage of plants that did not drop ears prior toharvest.

D/T=DROUGHT TOLERANCE. This represents a 1–9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance.

EAR HT=EAR HEIGHT. The ear height is a measure from the ground to thehighest placed developed ear node attachment and is measured in inches.

EAR MLD=General Ear Mold. Visual rating (1–9 score) where a “1” is verysusceptible and a “9” is very resistant. This is based on overall ratingfor ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold.

EAR SZ=EAR SIZE. A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

EBTSTK=EARLY BRITTLE STALK. A count of the number of “snapped” plantsper plot following severe winds when the corn plant is experiencing veryrapid vegetative growth in the V5–V8 stage. Expressed as percent ofplants that did not snap.

ECB 1 LF=EUROPEAN CORN BORER FIRST GENERATION LEAF FEEDING (Ostrinianubilalis). A 1 to 9 visual rating indicating the resistance topreflowering leaf feeding by first generation European Corn Borer. Ahigher score indicates a higher resistance.

ECB 2IT=EUROPEAN CORN BORER SECOND GENERATION INCHES OF TUNNELING(Ostrinia nubilalis). Average inches of tunneling per plant in thestalk.

ECB 2SC=EUROPEAN CORN BORER SECOND GENERATION (Ostrinia nubilalis). A 1to 9 visual rating indicating post flowering degree of stalk breakageand other evidence of feeding by European Corn Borer, Second Generation.A higher score indicates a higher resistance.

ECB DPE=EUROPEAN CORN BORER DROPPED EARS (Ostrinia nubilalis). Droppedears due to European Corn Borer. Percentage of plants that did not dropears under second generation corn borer infestation.

EGR WTH=EARLY GROWTH. This is a measure of the relative height and sizeof a corn seedling at the 2–4 leaf stage of growth. This is a visualrating (1 to 9), with 1 being weak or slow growth, 5 being averagegrowth and 9 being strong growth. Taller plants, wider leaves, moregreen mass and darker color constitute higher score.

ERTLDG=EARLY ROOT LODGING. Count for severity of plants that lean from avertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as percent of plantsnot lodged.

ERTLSC=EARLY ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as a 1 to 9 scorewith 9 being no lodging.

EST CNT=EARLY STAND COUNT. This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on perplot basis for the inbred or hybrid.

EYE SPT=Eye Spot (Kabatiella zeae or Aureobasidium zeae). A 1 to 9visual rating indicating the resistance to Eye Spot. A higher scoreindicates a higher resistance.

FUS ERS=FUSARIUM EAR ROT SCORE (Fusarium moniliforme or Fusariumsubglutinans). A 1 to 9 visual rating indicating the resistance toFusarium ear rot. A higher score indicates a higher resistance.

GDU=Growing Degree Units. Using the Barger Heat Unit Theory, whichassumes that maize growth occurs in the temperature range 50° F. –86° F.and that temperatures outside this range slow down growth; the maximumdaily heat unit accumulation is 36 and the minimum daily heat unitaccumulation is 0. The seasonal accumulation of GDU is a major factor indetermining maturity zones.

GDU SHD=GDU TO SHED. The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated by the Barger Method,where the heat units for a 24-hour period are:

${GDU} = {\frac{( {{Max}.\;{temp}.{+ {{Min}.\;{temp}.}}} )}{2} - 50}$

The highest maximum temperature used is 86° F. and the lowest minimumtemperature used is 50° F. For each inbred or hybrid it takes a certainnumber of GDUs to reach various stages of plant development.

GDU SLK=GDU TO SILK. The number of growing degree units required for aninbred line or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDU SHD definition.

GIB ERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae). A 1 to 9visual rating indicating the resistance to Gibberella Ear Rot. A higherscore indicates a higher resistance.

GIBROT=GIBBERELLA STALK ROT SCORE. Score of stalk rot severity due toGibberella (Gibberella zeae). Expressed as a 1 to 9 score with 9 beinghighly resistant.

GLF SPT=Gray Leaf Spot (Cercospora zeae-maydis). A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance.

GOS WLT=Goss' Wilt (Corynebacterium nebraskense). A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance.

GRN APP=GRAIN APPEARANCE. This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. High scores indicate good grain quality.

H/POP=YIELD AT HIGH DENSITY. Yield ability at relatively high plantdensities on 1–9 relative rating system with a higher number indicatingthe hybrid responds well to high plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to increased plant density.

HC BLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporiumcarbonum). A 1 to 9 visual rating indicating the resistance toHelminthosporium infection. A higher score indicates a higherresistance.

HD SMT=HEAD SMUT (Sphacelotheca reiliana). This score indicates thepercentage of plants not infected.

HSK CVR=HUSK COVER. A 1 to 9 score based on performance relative to keychecks, with a score of 1 indicating very short husks, tip of ear andkernels showing; 5 is intermediate coverage of the ear under mostconditions, sometimes with thin husk; and a 9 has husks extending andclosed beyond the tip of the ear. Scoring can best be done nearphysiological maturity stage or any time during dry down untilharvested.

INC D/A=GROSS INCOME (DOLLARS PER ACRE). Relative income per acreassuming drying costs of two cents per point above 15.5 percent harvestmoisture and current market price per bushel.

INCOME/ACRE. Income advantage of hybrid to be patented over other hybridon per acre basis.

INC ADV=GROSS INCOME ADVANTAGE. GROSS INCOME advantage of variety #1over variety #2.

KSZ DCD=KERNEL SIZE DISCARD. The percent of discard seed; calculated asthe sum of discarded tip kernels and extra large kernels.

L/POP=YIELD AT LOW DENSITY. Yield ability at relatively low plantdensities on a 1–9 relative system with a higher number indicating thehybrid responds well to low plant densities for yield relative to otherhybrids. A 1, 5, and 9 would represent very poor, average, and very goodyield response, respectively, to low plant density.

LRTLDG=LATE ROOT LODGING. Count for severity of plants that lean from avertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed aspercent of plants not lodged.

LRTLSC=LATE ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. his lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed asa 1 to 9 score with 9 being no lodging.

MDM CPX=MAIZE DWARF MOSAIC COMPLEX (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance.

MST=HARVEST MOISTURE. The moisture is the actual percentage moisture ofthe grain at harvest.

MST ADV=MOISTURE ADVANTAGE. The moisture advantage of variety #1 overvariety #2 as calculated by: MOISTURE of variety #2−MOISTURE of variety#1=MOISTURE ADVANTAGE of variety #1.

NLF BLT=Northern Leaf Blight (Helminthosporium turcicum or Exserohilumturcicum). A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance.

OILT=GRAIN OIL. Absolute value of oil content of the kernel as predictedby Near-Infrared Transmittance and expressed as a percent of dry matter.

PLT HT=PLANT HEIGHT. This is a measure of the height of the plant fromthe ground to the tip of the tassel in inches.

POL SC=POLLEN SCORE. A 1 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POL WT=POLLEN WEIGHT. This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

POP K/A=PLANT POPULATIONS. Measured as 1000s per acre.

POP ADV=PLANT POPULATION ADVANTAGE. The plant population advantage ofvariety #1 over variety #2 as calculated by PLANT POPULATION of variety#2−PLANT POPULATION of variety #1=PLANT POPULATION ADVANTAGE of variety#1.

PRM=PREDICTED RELATIVE MATURITY. This trait, predicted relativematurity, is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizes standardlinear regression analyses and is also referred to as the ComparativeRelative Maturity Rating System that is similar to the MinnesotaRelative Maturity Rating System.

PRM SHD=A relative measure of the growing degree units (GDU) required toreach 50% pollen shed. Relative values are predicted values from thelinear regression of observed GDU's on relative maturity of commercialchecks.

PROT=GRAIN PROTEIN. Absolute value of protein content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

RT LDG=ROOT LODGING. Root lodging is the percentage of plants that donot root lodge; plants that lean from the vertical axis at anapproximately 30° angle or greater would be counted as root lodged.

RTL ADV=ROOT LODGING ADVANTAGE. The root lodging advantage of variety #1over variety #2.

SCT GRN=SCATTER GRAIN. A 1 to 9 visual rating indicating the amount ofscatter grain (lack of pollination or kernel abortion) on the ear. Thehigher the score the less scatter grain.

SDG VGR=SEEDLING VIGOR. This is the visual rating (1 to 9) of the amountof vegetative growth after emergence at the seedling stage(approximately five leaves). A higher score indicates better vigor.

SEL IND=SELECTION INDEX. The selection index gives a single measure ofthe hybrid's worth based on information for up to five traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SLF BLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis). A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance.

SOU RST=SOUTHERN RUST (Puccinia polysora). A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance.

STA GRN=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STD ADV=STALK STANDING ADVANTAGE. The advantage of variety #1 overvariety #2 for the trait STK CNT.

STK CNT=NUMBER OF PLANTS. This is the final stand or number of plantsper plot.

STK LDG=STALK LODGING. This is the percentage of plants that did notstalk lodge (stalk breakage) as measured by either natural lodging orpushing the stalks and determining the percentage of plants that breakbelow the ear.

STKLDL=LATE SEASON STALK LODGING. A plant is considered as stalk lodgedif the stalk is broken or crimped between the ear and the ground. Thiscan be caused by any or a combination of the following: strong windslate in the season, disease pressure within the stalks, ECB damage orgenetically weak stalks. This trait should be taken when the grainmoisture content of the experiment is between 15% to 18%. Expressed aspercent of plants that did not stalk lodge.

STKLDS=REGULAR STALK LODGING SCORE. A plant is considered as stalklodged if the stalk is broken or crimped between the ear and the ground.This can be caused by any or a combination of the following: strongwinds late in the season, disease pressure within the stalks, ECB damageor genetically weak stalks. This trait should be taken just prior to orat harvest. Expressed on a 1 to 9 scale with 9 being no lodging.

STRT=GRAIN STARCH. Absolute value of starch content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

STW WLT=Stewart's Wilt (Erwinia stewartil). A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance.

TAS BLS=TASSEL BLAST. A 1 to 9 visual rating was used to measure thedegree of blasting (necrosis due to heat stress) of the tassel at thetime of flowering. A 1 would indicate a very high level of blasting attime of flowering, while a 9 would have no tassel blasting.

TAS SZ=TASSEL SIZE. A 1 to 9 visual rating was used to indicate therelative size of the tassel. The higher the rating the larger thetassel.

TAS WT=TASSEL WEIGHT. This is the average weight of a tassel (grams)just prior to pollen shed.

TEX EAR=EAR TEXTURE. A 1 to 9 visual rating was used to indicate therelative hardness (smoothness of crown) of mature grain. A 1 would bevery soft (extreme dent) while a 9 would be very hard (flinty or verysmooth crown).

TILLER=TILLERS. A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot.

TST WT=TEST WEIGHT (UNADJUSTED). The measure of the weight of the grainin pounds for a given volume (bushel).

TST WTA=TEST WEIGHT ADJUSTED. The measure of the weight of the grain inpounds for a given volume (bushel) adjusted for 15.5 percent moisture.

TSW ADV=TEST WEIGHT ADVANTAGE. The test weight advantage of variety #1over variety #2.

WIN M %=PERCENT MOISTURE WINS.

WIN Y %=PERCENT YIELD WINS.

YLD=YIELD. It is the same as BU ACR ABS.

YLD ADV=YIELD ADVANTAGE. The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1−YIELD variety #2=yieldadvantage of variety #1.

YLD SC=YIELD SCORE. A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

DETAILED DESCRIPTION OF THE INVENTION

Inbred maize lines are typically developed for use in the production ofhybrid maize lines. Inbred maize lines need to be highly homogeneous,homozygous and reproducible to be useful as parents of commercialhybrids. There are many analytical methods available to determine thehomozygotic and phenotypic stability of these inbred lines.

The oldest and most traditional method of analysis is the observation ofphenotypic traits. The data is usually collected in field experimentsover the life of the maize plants to be examined. Phenotypiccharacteristics most often observed are for traits associated with plantmorphology, ear and kernel morphology, insect and disease resistance,maturity, and yield.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), and Simple SequenceRepeats (SSRs) which are also referred to as Microsatellites.

The most widely used of these laboratory techniques are IsozymeElectrophoresis and RFLPs as discussed in Lee, M., “Inbred Lines ofMaize and Their Molecular Markers,” The Maize Handbook,(Springer-Verlag, New York, Inc. 1994, at 423–432) incorporated hereinby reference. Isozyme Electrophoresis is a useful tool in determininggenetic composition, although it has relatively low number of availablemarkers and the low number of allelic variants among maize inbreds.RFLPs have the advantage of revealing an exceptionally high degree ofallelic variation in maize and the number of available markers is almostlimitless.

Maize RFLP linkage maps have been rapidly constructed and widelyimplemented in genetic studies. One such study is described inBoppenmaier, et al., “Comparisons among strains of inbreds for RFLPS”,Maize Genetics Cooperative Newsletter, 65:1991, pg. 90, is incorporatedherein by reference. This study used 101 RFLP markers to analyze thepatterns of 2 to 3 different deposits each of five different inbredlines. The inbred lines had been selfed from 9 to 12 times before beingadopted into 2 to 3 different breeding programs. It was results fromthese 2 to 3 different breeding programs that supplied the differentdeposits for analysis. These five lines were maintained in the separatebreeding programs by selfing or sibbing and rogueing off-type plants foran additional one to eight generations. After the RFLP analysis wascompleted, it was determined the five lines showed 0–2% residualheterozygosity. Although this was a relatively small study, it can beseen using RFLPs that the lines had been highly homozygous prior to theseparate strain maintenance.

Inbred maize line PH6WR is a yellow, dent maize inbred that is suited asa male for producing first generation F1 maize hybrids. Inbred maizeline PH6WR is best adapted to the Central Corn Belt and Western regionsof the United States and can be used to produce hybrids fromapproximately 111 relative maturity based on the Comparative RelativeMaturity Rating System for harvest moisture of grain. Inbred maize linePH6WR demonstrates high yield, below average barrenness, very good standestablishment and above average early growth as an inbred per se. Inhybrid combination, including for its area of adaptation, inbred PH6WRdemonstrates below average plant height, very good resistance to brittlestalk and above average late season stalk strength.

The inbred has shown uniformity and stability within the limits ofenvironmental influence for all the traits as described in the VarietyDescription Information (Table 1) that follows. The inbred has beenself-pollinated and ear-rowed a sufficient number of generations withcareful attention paid to uniformity of plant type to ensure thehomozygosity and phenotypic stability necessary to use in commercialproduction. The line has been increased both by hand and in isolatedfields with continued observation for uniformity. No variant traits havebeen observed or are expected in PH6WR.

Inbred maize line PH6WR, being substantially homozygous, can bereproduced by planting seeds of the line, growing the resulting maizeplants under self-pollinating or sib-pollinating conditions withadequate isolation, and harvesting the resulting seed, using techniquesfamiliar to the agricultural arts.

TABLE 1 VARIETY DESCRIPTION INFORMATION VARIETY = PH6WR  1. TYPE:(describe intermediate types in Comments section): 2 1 = Sweet 2 = Dent3 = Flint 4 = Flour 5 = Pop 6 = Ornamental  2. MATURITY: DAYS HEAT UNITS076 1,440.0 From emergence to 50% of plants in silk 076 1,440.0 Fromemergence to 50% of plants in pollen 003 0,071.0 From 10% to 90% pollenshed From 50% silk to harvest at 25% moisture Standard Sample  3. PLANT:Deviation Size 0,207.7 cm Plant Height (to tassel tip) 9.29 15 0,085.3cm Ear Height (to base of top ear node) 8.02 15 0,015.9 cm Length of TopEar Internode 0.46 15 0.0 Average Number of Tillers 0.01 3 0.9 AverageNumber of Ears per Stalk 0.13 3 5.0 Anthocyanin of Brace Roots: 1 =Absent 2 = Faint 3 = Moderate 4 = Dark 5 = Very Dark Standard Sample  4.LEAF: Deviation Size 010.4 cm Width of Ear Node Leaf 0.20 15 078.9 cmLength of Ear Node Leaf 1.60 15 05.7 Number of leaves above top ear 0.3115 014.4 Degrees Leaf Angle (measure from 2nd leaf 3.62 15 above ear atanthesis to stalk above leaf) 03 Leaf Color Dark Green (Munsell code)5GY34 1.0 Leaf Sheath Pubescence (Rate on scale from 1 = none to 9 =like peach fuzz) Marginal Waves (Rate on scale from 1 = none to 9 =many) Longitudinal Creases (Rate on scale from 1 = none to 9 = many)Standard Sample  5. TASSEL: Deviation Size 10.5 Number of PrimaryLateral Branches 1.21 15 030.9 Branch Angle from Central Spike 3.60 1552.2 cm Tassel Length (from top leaf collar to tassel tip) 3.33 15 6.3Pollen Shed (rate on scale from 0 = male sterile to 9 = heavy shed) 14Anther Color Red (Munsell code) 10RP38 01 Glume Color Light Green(Munsell code) 5GY66 1.0 Bar Glumes (Glume Bands): 1 = Absent 2 =Present 19 cm Peduncle Length (cm. from top leaf to basal branches)  6a.EAR (Unhusked Data): 11 Silk Color (3 days after emergence) Pink(Munsell code) 5R58 3 Fresh Husk Color (25 days after 50% silking) DarkGreen (Munsell code) 5GY56 21 Dry Husk Color (65 days after 50% silking)Buff (Munsell code) 5Y92 3 Position of Ear at Dry Husk Stage: 1 =Upright 2 = Horizontal 3 = Pendant Pendant 5 Husk Tightness (Rate ofScale from 1 = very loose to 9 = very tight) 2 Husk Extension (atharvest): 1 = Short (ears exposed) 2 = Medium (<8 cm) Medium 3 = Long(8–10 cm beyond ear tip) 4 = Very Long (>10 cm) Standard Sample  6b. EAR(Husked Ear Data): Deviation Size 17 cm Ear Length 0.58 15 41 mm EarDiameter at mid-point 1.15 15 116 gm Ear Weight 23.80 15 17 Number ofKernel Rows 0.58 15 2 Kernel Rows: 1 = Indistinct 2 = Distinct Distinct2 Row Alignment: 1 = Straight 2 = Slightly Curved 3 = Spiral SlightlyCurved 7 cm Shank Length 0.58 15 2 Ear Taper: 1 = Slight 2 = Average 3 =Extreme Average Standard Sample  7. KERNEL (Dried): Deviation Size 11 mmKernel Length 0.58 15 8 mm Kernel Width 0.58 15 5 mm Kernel Thickness0.00 15 22 % Round Kernels (Shape Grade) 9.50 3 1 Aleurone ColorPattern: 1 = Homozygous 2 = Segregating Homozygous 7 Aluerone ColorYellow (Munsell code) 10YR714 7 Hard Endosperm Color Yellow (Munsellcode) 1.25Y714 3 Endosperm Type: Normal Starch 1 = Sweet (Su1) 2 = ExtraSweet (sh2) 3 = Normal Starch 4 = High Amylose Starch 5 = Waxy Starch 6= High Protein 7 = High Lysine 8 = Super Sweet (se) 9 = High Oil 10 =Other     25 gm Weight per 100 Kernels (unsized sample) 4.51 3 StandardSample  8. COB: Deviation Size 23 mm Cob Diameter at mid-point 0.58 1514 Cob Color Red (Munsell code) 10R68  9. DISEASE RESISTANCE (Rate from1 (most susceptible) to 9 (most resistant); leave  blank if not tested;leave Race or Strain Options blank if polygenic): A. Leaf Blights,Wilts, and Local Infection Diseases Anthracnose Leaf Blight(Colletotrichum graminicola) 5 Common Rust (Puccinia sorghi) Common Smut(Ustilago maydis) Eyespot (Kabatiella zeae) Goss's Wilt (Clavibactermichiganense spp. nebraskense) 5 Gray Leaf Spot (Cercospora zeae-maydis)Helminthosporium Leaf Spot (Bipolaris zeicola)  Race   7 Northern LeafBlight (Exserohilum turcicum)  Race   6 Southern Leaf Blight (Bipolarismaydis)  Race   Southern Rust (Puccinia polysora) 5 Stewart's Wilt(Erwinia stewartii) Other (Specify)    B. Systemic Diseases Corn LethalNecrosis (MCMV and MDMV) Head Smut (Sphacelotheca reiliana) MaizeChlorotic Dwarf Virus (MDV) Maize Chlorotic Mottle Virus (MCMV) 3 MaizeDwarf Mosaic Virus (MDMV) Sorghum Downy Mildew of Corn(Peronosclerospora sorghi) Other (Specify)    C. Stalk Rots 5Anthracnose Stalk Rot (Colletorichum graminicola) Diplodia Stalk Rot(Stenocarpella maydis) Fusarium Stalk Rot (Fusarium moniliforme)Gibberella Stalk Rot (Gibberella zeae) Other (Specify)    D. Ear andKernel Rots Aspergillus Ear and Kernel Rot (Aspergillus flavus) 5Diplodia Ear Rot (Stenocarpella maydis) 7 Fusarium Ear and Kernel Rot(Fusarium moniliforme) Gibberella Ear Rot (Gibberella zeae) Other(Specify)    10. INSECT RESISTANCE (Rate from 1 (most susceptible) to 9(most resistant);  (leave blank if not tested): Banks grass Mite(Oligonychus pratensis) Corn Worm (Helicoverpa zea) Leaf Feeding SilkFeeding mg larval wt. Ear Damage Corn Leaf Aphid (Rhopalosiphum maidis)Corn Sap Beetle (Carpophilus dimidiatus European Corn Borer (Ostrinianubilalis) 1st Generation (Typically Whorl Leaf Feeding) 2nd Generation(Typically Leaf Sheath-Collar Feeding) Stalk Tunneling cm tunneled/plantFall Armyworm (Spodoptera fruqiperda) Leaf Feeding Silk Feeding mglarval wt. Maize Weevil (Sitophilus zeamaize Northern Rootworm(Diabrotica barberi) Southern Rootworm (Diabrotica undecimpunctata)Southwestern Corn Borer (Diatreaea grandiosella) Leaf Feeding StalkTunneling cm tunneled/plant Two-spotted Spider Mite (Tetranychusurticae) Western Rootworm (Diabrotica virgifrea virgifera) Other(Specify)    11. AGRONOMIC TRAITS: 5 Staygreen (at 65 days afteranthesis) (Rate on a scale from 1 = worst to 9 = excellent) % DroppedEars (at 65 days after anthesis) % Pre-anthesis Brittle Snapping %Pre-anthesis Root Lodging 1.3 Post-anthesis Root Lodging (at 65 daysafter anthesis) 5,165 Kg/ha Yield (at 12–13% grain moisture) *Ininterpreting the foregoing color designations, reference may be made tothe Munsell Glossy Book of Color, a standard color reference.

Further Embodiments of the Invention

This invention also is directed to methods for producing a maize plantby crossing a first parent maize plant with a second parent maize plantwherein either the first or second parent maize plant is an inbred maizeplant of the line PH6WR. Further, both first and second parent maizeplants can come from the inbred maize line PH6WR. Still further, thisinvention also is directed to methods for producing an inbred maize linePH6WR-derived maize plant by crossing inbred maize line PH6WR with asecond maize plant and growing the progeny seed, and repeating thecrossing and growing steps with the inbred maize line PH6WR-derivedplant from 0 to 5 times. Thus, any such methods using the inbred maizeline PH6WR are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing inbred maize line PH6WR as a parent are within the scope of thisinvention, including plants derived from inbred maize line PH6WR.Advantageously, the inbred maize line is used in crosses with other,different, maize inbreds to produce first generation (F₁) maize hybridseeds and plants with superior characteristics.

A further embodiment of the invention is a single gene conversion orintrogression of the maize plant disclosed herein in which the gene orgenes of interest (encoding the desired trait) are introduced throughtraditional (non-transformation) breeding techniques, such asbackcrossing (Hallauer et al, 1988). One or more genes may be introducedusing these techniques. Desired traits transferred through this processinclude, but are not limited to, waxy starch, nutritional enhancements,industrial enhancements, disease resistance, insect resistance,herbicide resistance and yield enhancements. The gene of interest istransferred from the donor parent to the recurrent parent, in this case,the maize plant disclosed herein. These single gene traits may resultfrom either the transfer of a dominant allele or a recessive allele.Selection of progeny containing the trait of interest is done by directselection for a trait associated with a dominant allele. Selection ofprogeny for a trait that is transferred via a recessive allele, such asthe waxy starch characteristic, requires growing and selfing the firstbackcross to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the gene of interest.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, seeds, flowers,kernels, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk and the like.

Duncan, Williams, Zehr, and Widholm, Planta (1985) 165:322–332 reflectsthat 97% of the plants cultured that produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262–265 reports several media additions that enhance regenerability ofcallus of two inbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao, et al., Maize Genetics CooperationNewsletter, 60:64–65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345–347(1987) indicates somatic embryogenesis from the tissue cultures of maizeleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of maize is described in European Patent Application,publication 160,390, incorporated herein by reference. Maize tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 1982, at367–372) and in Duncan, et al., “The Production of Callus Capable ofPlant Regeneration from Immature Embryos of Numerous Zea MaysGenotypes,” 165 Planta 322–332 (1985). Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce maize plants having the physiological and morphologicalcharacteristics of inbred line PH6WR.

The utility of inbred maize line PH6WR also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with PH6WR may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

Transformation of Maize

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native orendogenous genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreign,additional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred maize line PH6WR.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used, alone or incombination with other plasmids, to provide transformed maize plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the maize plant(s).

Expression Vectors For Maize Transformation

Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e. inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase 11 (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80: 4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol, 5: 299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210: 86 (1987), Svab etal., Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317: 741–744 (1985), Gordon-Kamm et al., Plant Cell 2: 603–618(1990) and Stalker et al., Science 242: 419–423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8: 643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5: 387 (1987)., Teeri et al., EMBO J. 8: 343(1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247: 449(1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 2908, Imagene Green™, p. 1–4 (1993) and Naleway etal., J. Cell Biol. 115: 151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263: 802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissues arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inmaize. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in maize. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol.22: 361–366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al. PNAS 90: 4567–4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227: 229–237 (1991) and Gatz et al.,Mol. Gen. Genetics 243: 32–38 (1994)) or Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genet. 227: 229–237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inmaize or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in maize.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313: 810–812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163–171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12: 619–632 (1989) andChristensen et al., Plant Mol. Biol. 18: 675–689 (1992)): pEMU (Last etal., Theor. Appl. Genet. 81: 581–588 (1991)); MAS (Velten et al., EMBOJ. 3: 2723–2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genet. 231: 276–285 (1992) and Atanassova et al., Plant Journal 2 (3):291–300 (1992)).

The ALS promoter, a Xbal/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence that has substantial sequencesimilarity to said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin maize. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in maize. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23: 476–482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320–3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11): 2723–2729(1985) and Timko et al., Nature 318: 579–582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genet. 217:240–245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genet.224: 161–168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6: 217–224 (1993).

Signal Sequences For Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art. See, for example, Becker etal., Plant Mol. Biol.20: 49 (1992), Close, P. S., Master's Thesis, IowaState University (1993), Knox, C., et al., “Structure and Organizationof Two Divergent Alpha-Amylase Genes From Barley”, Plant Mol. Biol. 9:3–17 (1987), Lerner et al., Plant Physiol.91: 124–129 (1989), Fontes etal., Plant Cell 3: 483–496 (1991), Matsuoka et al., Proc. Natl. Acad.Sci. 88: 834 (1991), Gould et al., J. Cell Biol 108: 1657 (1989),Creissen et al., Plant J. 2: 129 (1991), Kalderon, D., Robers, B.,Richardson, W., and Smith A., “A short amino acid sequence able tospecify nuclear location”, Cell 39: 499–509 (1984), Stiefel, V.,Ruiz-Avila, L., Raz R., Valles M., Gomez J., Pages M.,Martinez-Izquierdo J., Ludevid M., Landale J., Nelson T., andPuigdomenech P., “Expression of a maize cell wall hydroxyproline-richglycoprotein gene in early leaf and root vascular differentiation”,Plant Cell 2: 785–793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92–6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is maize. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, and Simple Sequence Repeats (SSR) which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, METHODSIN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 269–284 (CRC Press, BocaRaton, 1993). Map information concerning chromosomal location is usefulfor proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below.

1. Genes That Confer Resistance To Pests or Disease And That Encode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Rockville,Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

(C) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(D) A vitamin-binding protein such as avidin. See PCT applicationUS93/06487 the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

(E) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huubet al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor 1), and Sumitani et al., Biosci.Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

(H) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

(I) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(K) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(L) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

(M) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(P) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2: 367 (1992).

(R) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance To A Herbicide. For Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European Patent Application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EuropeanApplication No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes That Confer Or Contribute To A Value-Added Trait, Such As:

(A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89: 2624 (1992).

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35: 383 (1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102: 1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Maize Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67–88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89–119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant. Sci. 10: 1 (1991). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber et al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8: 238 (1989). See also, U.S. Pat. No. 5,591,616, issued Jan. 7,1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and maize. Hieiet al., The Plant Journal 6: 271–282 (1994); U.S. Pat. No. 5,591,616,issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5: 27 (1987), Sanford, J. C., Trends Biotech. 6: 299(1988), Klein et al., Bio/Technology 6: 559–563 (1988), Sanford, J. C.,Physiol Plant 79: 206 (1990), Klein et al., Biotechnology 10: 268(1992). In maize, several target tissues can be bombarded withDNA-coated microprojectiles in order to produce transgenic plants,including, for example, callus (Type I or Type II), immature embryos,and meristematic tissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199: 161 (1985) and Draper et al., Plant Cell Physiol. 23: 451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4: 1495–1505 (1992) and Spencer et al.,Plant Mol. Biol. 24: 51–61 (1994).

Following transformation of maize target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art. Forexample, transformed maize immature embryos.

The foregoing methods for transformation would typically be used forproducing transgenic inbred lines. Transgenic inbred lines could then becrossed, with another (non-transformed or transformed) inbred line, inorder to produce a transgenic hybrid maize plant. Alternatively, agenetic trait which has been engineered into a particular maize lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-elite lineinto an elite line, or from a hybrid maize plant containing a foreigngene in its genome into a line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

INDUSTRIAL APPLICABILITY

Maize is used as human food, livestock feed, and as raw material inindustry. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.The principal products of maize dry milling are grits, meal and flour.The maize wet-milling industry can provide maize starch, maize syrups,and dextrose for food use. Maize oil is recovered from maize germ, whichis a by-product of both dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry: forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred maize line PH6WR, the plant produced from the inbredseed, the hybrid maize plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid maize plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

Performance Examples of PH6WR

In the examples that follow, the traits and characteristics of inbredmaize line PH6WR are given as a line. The data collected on inbred maizeline PH6WR is presented for the key characteristics and traits.

Inbred Comparisons

The results in Table 2A compare inbred PH6WR to inbred PHR03. InbredPH6WR produces an above average and significantly higher yield withsignificantly lower harvest moisture than inbred PHR03. Inbred PH6WRshows above average and significantly better early growth and asignificantly better early stand count than inbred PHR03. Inbred PH6WRflowers (GDU SHD and GDU SLK) significantly earlier than inbred PHR03.Inbred PH6WR presents a significantly shorter plant with significantlylower ear placement than inbred PHR03. Inbred PH6WR shows few barrenplants.

The results in Table 2B compare inbred PH6WR to inbred PHPP8. InbredPH6WR produces an above average yield. Inbred PH6WR shows above averageearly growth and a significantly better early stand count than inbredPHPP8. Inbred PH6WR presents a significantly shorter plant than inbredPHPP8. Inbred PH6WR shows few barren plants.

Inbred By Tester Comparisons

The results in Table 3A compare the inbred PH6WR and inbred PH1B5, wheneach inbred is crossed to the same tester lines. The PH6WR hybridsdemonstrate good yield and significantly lower harvest moisture of grainthan the PH1B5 hybrids. The PH6WR hybrids present a below average plantheight. The PH6WR hybrids show above average early stand establishmentand significantly better final stand counts than the PH1B5 hybrids. ThePH6WR hybrids show above average resistance to brittle stalk.

The results in Table 3B compare the inbred PH6WR and inbred PH2MW, wheneach inbred is crossed to the same tester lines. The PH6WR hybridsdemonstrate good yield and significantly lower harvest moisture of grainthan the PH2MW hybrids. The PH6WR hybrids present a significantlyshorter plant than the PH2MW hybrids.

Hybrid Comparisons

The results in Table 4 compare inbred PH09B crossed to inbred PH6WR andinbred PH09B crossed to inbred PH1CA. The results show the PH09B/PH6WRhybrid to demonstrate good yields. The PH09B/PH6WR hybrid shows aboveaverage early stand count and flowers (GDU SHD and GDU SLK)significantly earlier than the PH09B/PH1CA hybrid. The PH09B/PH6WRhybrid presents a significantly lower plant height than the PH09B/PH1CAhybrid. The PH09B/PH6WR hybrid shows above average and significantlybetter artificial brittle stalk resistance than the PH09B/PH1CA hybridand shows a significantly better regular stalk lodging score than thePH09B/PH1CA hybrid.

TABLE 2A PAIRED INBRED COMPARISON REPORT VARIETY #1 = PH6WR VARIETY #2 =PHR03 BU BU EGR EST TIL GDU GDU POL ACR ACR MST WTH CNT LER SHD SLK WTABS % MN ABS ABS ABS ABS ABS ABS % MN TOTAL SUM 1 91.7 135 18.9 5.6 21.86.4 146.6 149.2 131 2 75.5 111 21.3 5.2 20.0 4.5 152.2 155.0 103 LOCS 1010 11 27 26 23 64 64 8 REPS 10 10 11 27 26 23 64 64 16 DIFF 16.2 25 2.40.4 1.8 1.9 5.6 5.8 28 PR > T .010+ .010+ .049+ .048+ .029+ .571 .000#.000# .020+ POL TAS PLT EAR STA STK STK BRT SCT SC SZ HT HT GRN LDS LDGSTK GRN ABS ABS ABS ABS ABS ABS ABS ABS ABS TOTAL SUM 1 6.0 6.1 84.233.5 5.2 7.0 100.0 100.0 6.4 2 5.0 5.9 89.3 38.8 5.3 5.0 100.0 100.0 7.5LOCS 1 46 37 6 12 2 4 2 12 REPS 1 46 37 6 12 2 4 2 12 DIFF 1.0 0.2 5.15.3 0.1 2.0 0.0 0.0 1.1 PR > T .192 .000# .028+ .881 .500 .999 .999.012+ TEX EAR BAR GLF NLF SLF STW ANT HD EAR MLD PLT SPT BLT BLT WLT ROTSMT ABS % MN ABS ABS ABS ABS ABS ABS ABS TOTAL SUM 1 6.3 101 97.7 5.66.8 5.5 4.3 4.9 98.1 2 7.0 115 97.6 5.8 4.5 5.3 5.3 4.3 100.0 LOCS 3 1229 9 2 2 2 4 3 REPS 3 12 29 14 4 4 3 8 6 DIFF 0.7 14 0.1 0.2 2.3 0.3 1.00.6 1.9 PR > T .184 .031+ .919 .548 .070* .500 .500 .492 .423 MDM FUSDIP COM ECB ECB CLD KSZ CPX ERS ERS RST 1LF 2SC TST DCD ABS ABS ABS ABSABS ABS ABS ABS TOTAL SUM 1 3.0 6.9 5.5 5.6 7.5 1.5 91.8 4.8 2 3.3 7.04.8 5.6 7.0 4.0 84.4 6.0 LOCS 2 9 3 7 1 1 5 5 REPS 4 10 4 9 2 2 5 5 DIFF0.3 0.1 0.7 0.0 0.5 2.5 7.4 1.2 PR > T .500 .842 .423 .999 .209 .305 * =10% SIG + = 5% SIG # = 1% SIG

TABLE 2B PAIRED INBRED COMPARISON REPORT VARIETY #1 = PH6WR VARIETY #2 =PHPP8 BU BU EGR EST TIL GDU GDU POL ACR ACR MST WTH CNT LER SHD SLK WTABS % MN ABS ABS ABS ABS ABS ABS % MN TOTAL SUM 1 93.2 135 19.3 5.5 25.86.4 149.4 151.4 139 2 84.7 124 18.0 5.0 23.4 4.6 148.0 151.8 122 LOCS 99 10 11 5 14 29 29 6 REPS 9 9 10 11 5 14 29 29 12 DIFF 8.4 12 1.3 0.52.4 1.8 1.4 0.4 17 PR > T .130 .161 .142 .111 .016+ .596 .149 .712 .272TAS PLT EAR STA STK STK BRT SCT TEX SZ HT HT GRN LDS LDG STK GRN EAR ABSABS ABS ABS ABS ABS ABS ABS ABS TOTAL SUM 1 6.0 85.4 34.3 5.0 7.0 100.0100.0 6.5 6.5 2 6.5 91.6 36.0 2.8 7.0 100.0 99.0 7.8 6.0 LOCS 20 13 4 72 1 2 4 2 REPS 20 13 4 7 2 1 2 4 2 DIFF 0.6 6.2 1.8 2.2 0.0 0.0 1.0 1.30.5 PR > T .024+ .004# .422 .033+ .999 .500 .080* .500 EAR BAR GLF STWFUS DIP COM CLD KSZ MLD PLT SPT WLT ERS ERS RST TST DCD % MN ABS ABS ABSABS ABS ABS ABS ABS TOTAL SUM 1 108 97.1 5.3 4.0 8.5 9.0 4.5 91.8 4.8 2102 98.3 3.3 5.0 7.5 9.0 5.5 92.6 13.0 LOCS 3 13 3 1 2 1 2 5 5 REPS 3 133 1 2 1 2 5 5 DIFF 6 1.2 2.0 1.0 1.0 0.0 1.0 0.8 8.2 PR > T .423 .406.000# .000# .500 .528 .044+ * = 10% SIG + = 5% SIG # = 1% SIG

TABLE 3A Average Inbred By Tester Performance Comparing PH6WR To PH1B5Crossed To The Same Inbred Testers And Grown In The Same Experiments. BUBU SDG EST GDU GDU STK PLT ACR ACR MST VGR CNT SHD SLK CNT HT ABS % MN %MN % MN % MN % MN % MN % MN % MN TOTAL SUM REPS 137 137 139 15 15 30 19219 38 LOCS 137 137 139 15 15 30 19 219 38 PH6WR 178 99 97 94 101 100 99100 98 PH1B5 175 98 99 100 98 96 96 99 94 DIFF 3 1 2 5 2 3 3 1 4 PR > T0.20 0.29 0.00 0.22 0.29 0.00 0.00 0.01 0.00 EAR RT STA STK BRT SLF ANTHD HT LDG GRN LDG STK BLT ROT SMT CLN % MN % MN % MN % MN % MN ABS ABSABS ABS TOTAL SUM REPS 38 6 34 15 9 2 15 3 2 LOCS 38 6 34 15 9 2 15 3 2PH6WR 99 85 86 97 106 6 4 99 4 PH1B5 90 121 93 104 104 6 4 96 4 DIFF 936 7 7 1 0 0 2 0 PR > T 0.00 0.12 0.21 0.01 0.66 0.99 0.99 0.30 0.99 MDMFUS DIP COM ECB ECB DRP GLF NLF CPX ERS ERS RST 1LF 2SC EAR SPT BLT ABSABS ABS ABS ABS ABS % MN ABS ABS TOTAL SUM REPS 2 4 2 1 4 4 2 9 6 LOCS 24 2 1 4 4 2 9 6 PH6WR 3 4 5 5 5 4 99 5 5 PH1B5 2 4 5 4 4 4 100 4 5 DIFF1 0 1 1 1 0 1 1 0 PR > T 0.20 0.99 0.66 0.37 0.99 0.50 0.01 0.99 *PR > Tvalues are valid only for comparisons with Locs >= 10.

TABLE 3B Average Inbred By Tester Performance Comparing PH6WR To PH2MWCrossed To The Same Inbred Testers And Grown In The Same Experiments. BUBU SDG GDU STK PLT EAR STA STK GLF ACR ACR MST VGR SHD CNT HT HT GRN LDGSPT ABS % MN % MN % MN % MN % MN % MN % MN % MN % MN ABS TOTAL SUM REPS90 90 95 6 17 145 31 31 23 7 4 LOCS 90 90 95 6 17 145 31 31 23 7 2 PH6WR174 97 98 85 100 100 99 100 96 98 4 PH2MW 188 105 103 85 101 102 102 104102 102 4 DIFF 14 8 5 0 1 2 3 4 6 4 1 PR > T 0.00 0.00 0.00 0.99 0.000.02 0.01 0.06 0.45 0.57 0.50 *PR > T values are valid only forcomparisons with Locs >= 10.

TABLE 4 INBREDS IN HYBRID COMBINATION REPORT VARIETY #1 = PH09B/PH6WRVARIETY #2 = PH09B/PH1CA PRM BU BU TST GDU GDU PLT EAR PRM SHD ACR ACRMST WTA SHD SLK HT HT ABS ABS ABS % MN % MN ABS % MN % MN % MN % MNTOTAL SUM 1 112 112 177.9 100 97 56.8 99 100 97 100 2 112 112 177.5 9997 57.8 100 101 102 103 LOCS 3 1 116 116 118 92 26 23 29 29 REPS 3 1 126126 128 102 31 27 38 38 DIFF 0 1 0.4 0 0 1.0 1 1 4 3 PR > T .999 .830.999 .999 .000# .009# .042+ .000# .138 ERT RT LRT STK STK STK EBT ABTEGR STA LSC LDG LSC LDS LDG LDL STK STK WTH GRN ABS % MN ABS ABS % MN %MN % MN % MN % MN % MN TOTAL SUM 1 8.0 53 4.0 6.1 94 86 100 123 97 83 28.0 114 6.7 5.2 91 85 98 99 101 79 LOCS 2 2 3 41 9 20 4 8 18 30 REPS 2 43 42 9 38 6 27 18 37 DIFF 0.0 61 2.7 0.9 3 2 1 24 4 3 PR > T .999 .591.157 .030+ .530 .711 .681 .002# .440 .588 TST STK EST GLF NLF SLF ANT HDMDM WT CNT CNT SPT BLT BLT ROT SMT CLN CPX ABS % MN % MN ABS ABS ABS ABSABS ABS ABS TOTAL SUM 1 56.8 100 102 4.6 5.3 5.5 4.5 98.6 3.5 2.8 2 57.9101 100 5.1 5.0 5.0 3.8 91.1 4.1 2.3 LOCS 92 189 10 9 6 2 15 3 2 2 REPS102 277 13 15 10 4 24 6 8 4 DIFF 1.0 1 2 0.6 0.3 0.5 0.7 7.5 0.6 0.5PR > T .000# .086* .357 .003# .646 .000# .203 .137 .126 .500 FUS DIP COMECB ECB HSK SWB ERS ERS RST 1LF 2SC CVR PGR ABS ABS ABS ABS ABS ABS ABSTOTAL SUM 1 3.9 4.5 5.0 4.9 4.4 4.6 45.0 2 4.5 5.5 4.0 5.0 3.3 4.6 30.0LOCS 5 2 1 4 4 12 1 REPS 8 4 1 8 8 13 1 DIFF 0.6 1.0 1.0 0.1 1.1 0.015.0 PR > T .235 .000# .789 .058* .999 * = 10% SIG + = 5% SIG # = 1% SIG

Applicant has made a deposit of at least 2500 seeds of Inbred Maize LinePH6WR with the American Type Culture Collection (ATCC), Manassas, Va.20110 USA, ATCC Deposit No. PTA-4436. The seeds deposited with the ATCCon Jun. 4, 2002 were taken from the deposit maintained by PioneerHi-Bred International, Inc., 7100 NW 62nd Avenue, Johnston, Iowa50131-1000 since prior to the filing date of this application. Access tothis deposit will be available during the pendency of the application tothe Commissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant will make the deposit availableto the public pursuant to 37 C.F.R. § 1.808. This deposit of the InbredMaize Line PH6WR will be maintained in the ATCC depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicant has satisfied all the requirements of 37 C.F.R.§§ 1.801–1.809, including providing an indication of the viability ofthe sample upon deposit. Applicant has no authority to waive anyrestrictions imposed by law on the transfer of biological material orits transportation in commerce. Applicant does not waive anyinfringement of his rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq). U.S. Plant VarietyProtection of Inbred Maize Line PH6WR has been issued under PVP No.200100229. Unauthorized seed multiplication prohibited. U.S. ProtectedVariety.

1. A seed comprising at least one set of the chromosomes of maize inbredline PH6WR, representative seed of said line having been deposited underATCC Accession No. PTA-4436.
 2. A maize plant produced by growing theseed of claim
 1. 3. A maize plant part of the maize plant of claim
 2. 4.An F1 hybrid maize seed produced by crossing a plant of maize inbredline designated PH6WR, representative seed of said line having beendeposited under ATCC Accession No. PTA-4436, with a different maizeplant and harvesting the resultant F1 hybrid maize seed, wherein said F1hybrid maize seed comprises two sets of chromosomes and one set of thechromosomes is the same as maize inbred line PH6WR.
 5. A maize plantproduced by growing the F1 hybrid maize seed of claim
 4. 6. A maizeplant part of the maize plant of claim
 5. 7. An F1 hybrid maize seedcomprising an inbred maize plant cell of inbred maize line PH6WR,representative seed of said line having been deposited under ATCCAccession No. PTA-4436.
 8. A maize plant produced by growing the F1hybrid maize seed of claim
 7. 9. The F1 hybrid maize seed of claim 7wherein the inbred maize plant cell comprises two sets of chromosomes ofmaize inbred line PH6WR.
 10. A maize plant produced by growing the F1hybrid maize seed of claim
 9. 11. A maize plant having all thephysiological and morphological characteristics of inbred line PH6WR,wherein a sample of the seed of inbred line PH6WR was deposited underATCC Accession Number PTA-4436.
 12. A process of producing maize seed,comprising crossing a first parent maize plant with a second parentmaize plant, wherein one or both of the first or the second parent maizeplants is the plant of claim 11, wherein seed is allowed to form. 13.The maize seed produced by the process of claim
 12. 14. The maize seedof claim 13, wherein the maize seed is hybrid seed.
 15. A hybrid maizeplant, or its parts, produced by growing said hybrid seed of
 14. 16. Acell of the maize plant of claim
 11. 17. A seed comprising the cell ofclaim
 16. 18. The maize plant of claim 11, further comprising a genomecomprising a single gene conversion.
 19. The maize plant of claim 18,wherein the gene was stably inserted into a maize genome bytransformation.
 20. The maize plant of claim 18, wherein the gene isselected from the group consisting of a dominant allele and a recessiveallele.
 21. The maize plant of claim 18, wherein the gene confers atrait selected from the group consisting of herbicide tolerance; insectresistance; resistance to bacterial, fungal, or viral disease; waxystarch; male sterility and restoration of male fertility.
 22. The maizeplant of claim 11, wherein said plant further comprises a geneconferring male sterility.
 23. The maize plant of claim 11, wherein saidplant further comprises a transgene conferring a trait selected from thegroup consisting of male sterility, herbicide resistance, insectresistance, and disease resistance.
 24. A method of producing a maizeplant derived from the inbred line PH6WR, the method comprising thesteps of: (a) growing a progeny plant produced by crossing the plant ofclaim 11 with a second maize plant; (b) crossing the progeny plant withitself or a different plant to produce a seed of a progeny plant of asubsequent generation; (c) growing a progeny plant of a subsequentgeneration from said seed and crossing the progeny plant of a subsequentgeneration with itself or a different plant; and (d) repeating steps (b)and (c) for an additional 0–5 generations to produce a maize plantderived from the inbred line PH6WR.
 25. The method of claim 24, whereinthe maize plant derived from the inbred line PH6WR is an inbred maizeplant.
 26. The method of claim 25, further comprising the step ofcrossing the inbred maize plant derived from the inbred line PH6WR witha second, distinct inbred maize plant to produce an F1 hybrid maizeplant.
 27. A method for developing a maize plant in a maize plantbreeding program using plant breeding techniques comprising employing amaize plant, or its parts, as a source of plant breeding materialcomprising using the maize plant of claim 11, or parts thereof, as asource of said breeding material.
 28. The method for developing a maizeplant in a maize plant breeding program of 27 wherein plant breedingtechniques are selected from the group consisting of recurrentselection, backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.